Inhibition of mycobacterial arylamine N-acetyltransferase contributes to anti-mycobacterial activity of Warburgia salutaris

In this study, we show that extracts and a purified compound of Warburgia salutaris exhibit anti-mycobacterial activity against Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG Pasteur. The extracts did not inhibit growth of Escherichia coli and were not toxic to cultured mammalian macrophage cells at the concentrations at which anti-mycobacterial activity was observed. The extract and pure compound inhibited pure recombinant arylamine N-acetyltransferase (NAT), an enzyme involved in mycobacterial cell wall lipid synthesis. Moreover, neither extract nor pure compound inhibited growth of a strain of M. bovis BCG in which nat has been deleted suggesting that NAT may indeed be a target within the mycobacterial cell. The purified compound is a novel drimane sesquiterpenoid lactone, 11alpha-hydroxycinnamosmolide. These studies show that W. salutaris is a useful source of anti-tubercular compounds for further analysis and supports the hypothesis of a link between NAT inhibition and anti-mycobacterial activity.     

In vitro culture of two threatened South African medicinal trees – Ocotea bullata and Warburgia salutaris

The aim of the project was to develop micropropagation procedures for theheavily exploited and endangered South African trees black stinkwood (Ocotea bullata) and pepperbark (Warburgia salutaris) to facilitateconservation and reforestation. Both species are difficult to establish andgrow in tissue culture because of their high phenolic content. A protocol forthe establishment of explants in vitro was developed comprisingdecontamination, the application of antioxidants and cold treatment.     

Flavonol and drimane-type sesquiterpene glycosides of Warburgia stuhlmannii leaves

An investigation of methanolic extract of Warburgia stuhlmannii leaves has led to the isolation of two new drimane-type sesquiterpene glycosides characterized as mukaadial 6-O-beta-D-glucopyranoside, mukaadial 6-O-alpha-L-rhamnopyranoside together with two other novel flavonol glycosides identified as 3',5'-O-dimethylmyricetin 3-O-beta-D-2",3"-diacetylglucopyranoside and 3'-O-methylquercetin 3-O-beta-D-2",3",4"-triacetylglucopyranoside. The known compounds; mukaadial, deacetylugandensolide, quercetin, kaempferol, kaempferol 3-O-alpha-L-rhamnopyranoside, quercetin 3-O-beta-D-glucopyranoside, kaempferol 7-O-beta-D-glucopyranoside, myricetin 3-O-alpha-L-rhamnopyranoside, quercetin 3-O-alpha-L-rhamnopyranoside, quercetin 3-O-sophoroside and isorhamnetin 3-O-beta-D-glucopyranoside were also isolated from the same extract.     

Flavonol glycosides of Warburgia ugandensis leaves

Four new flavonol gycosides: kaempferide 3-O-beta-xylosyl (1-->2)-beta-glucoside, kaempferol 3-O-alpha-rhamnoside-7,4'-di-O-beta-galactoside, kaempferol 3,7,4'-tri-O-beta-glucoside and quercetin 3-O-[alpha-rhamnosyl (1-->6)] [beta-glucosyl (1-->2)]-beta-glucoside-7-O-alpha-rhamnoside, were characterized from a methanolic leaf extract of Warburgia ugandensis. The known flavonols: kaempferol, kaempferol 3-rhamnoside, kaempferol 3-rutinoside, myricetin, quercetin 3-rhamnoside, kaempferol 3-arabinoside, quercetin 3-glucoside, quercetin, kaempferol 3-rhamnoside-4'-galactoside, myricetin 3-galactoside and kaempferol 3-glucoside were also isolated. Structures were established by spectroscopic and chemical methods and by comparison with authentic samples.     

Alterations of chemoreceptor function in army worm larvae (Spodoptera exempta) by a plant-derived sesquiterpenoid and by sulfhydryl reagents

ABSTRACT. Warburganal, a sesquiterpene dialdehyde from Warburgia sp., strongly suppressed the feeding response of Spodoptera exempta larvae to sucrose-flavoured agar. Its effect on chemoreceptive sensilla of S. exempta was investigated. After a latent period (of up to an hour, depending on concentration), sensilla treated with warburganal produced transient bursting discharges in several receptor cells. Subsequently, receptor responses to sucrose or meso-inositol were reversibly inhibited. These effects were abolished by mixing warburganal with cysteine or dithiothreitol (though not with other amino acids), and were qualitatively similar to the effects of the sulfhydryl reagents PCMB and NEM. The possible mode of action by which warburganal may act as an –SH acceptor and thereby affect energy transduction in the chemoreceptors is discussed.     

Physiological activity of warburganal and its reactivity with sulfhydryl groups

Warburganal, a unique dialdehyde sesquiterpene isolated from East African Warburgia plants, showed a strong antifungal activity. However, this growth inhibition in Saccharomyces cerevisiae was reversed with L-cysteine. In addition, warburganal inhibited the alcoholic fermentation of S. cerevisiae while L-cysteine reversed this inhibition. When alcohol dehydrogenase, a sulfhydryl enzyme, was incubated with warburganal, the enzyme activity decreased with time. The decrease was more rapid at alkaline pH. L-Cysteine prevented this enzyme inhibition by warburganal but could not restore the enzyme activity lost already due to warburganal. Warburganal lost its characteristic ultraviolet absorption spectrum in the presence of L-cysteine. The change in absorbance was favored at alkaline pH, indicating Michael reaction type addition of L-cysteine to warburganal. Based on these observations, a variety of physiological activities due to warburganal appear to result from its irreversible reactivity with sulfhydryl groups.     

THE PHYSIOLOGICAL EFFECTS OF AQUEOUS SUSPENSIONS OF PLANT MOLLUSCICIDES ON HELISOMA DURYI (GASTROPODA: PLANORBIDAE)

The behavioural and physiological responses of Heltsoma duryito solutions of three South African medicinal, mollusricidalplants (Warburgia salutans, Gardenia thunbergia and Apodytesdimidiata subsp dimidiata) were investigated. Clinical signsof toxic-lty, oxygen consumption, heart rate and water uptakewere used to assess response to these mollus-cicides. All plantsproduced symptoms characteristic of the distress syndrome describedby Harry & Aldrich (1963). Haemolysis, however, occurredonly in response to extracts of G. thunbergia. Respiratory failurein snails exposed to the W. salutans and G. thunbergia did notappear to be the primary cause of mortality. All plants produceda significant lowering of heart rate at lethal concentrations.Water unbalance, which is a commonly suggested effect, did notoccur. The difficulty of inferring mode of action from observedeffects is emphasized. Although some responses are common tothe three plant mollusci-cides under investigation, the differencesclearly indicate the potential for alternative modes of action. (Received 14 November 1995; accepted 17 April 1996)     

Sesquiterpenes from Warburgia ugandensis and their antimycobacterial activity

The dichloromethane extract of the stem bark of Warburgia ugandensis afforded three new coloratane sesquiterpenes, namely: 6alpha,9alpha-dihydroxy-4(13),7-coloratadien-11,12-dial (1), 4(13),7-coloratadien-12,11-olide (2), and 7beta-hydroxy-4(13),8-coloratadien-11,12-olide (3), together with nine known sesquiterpenes, i.e., cinnamolide-3beta-acetate (4), muzigadial (5), muzigadiolide (6), 11alpha-hydroxymuzigadiolide (7), cinnamolide (8), 7alpha-hydroxy-8-drimen-11,12-olide (9), ugandensolide (10), mukaadial (11), ugandensidial (12), and linoleic acid (13). Their structures were assigned on the basis of 1D and 2D-NMR spectroscopic and GC-MS analysis. The compounds were examined for their antimycobacterial activity against Mycobacterium aurum, M. fortuitum, M. phlei and M. smegmatis; and the active constituents showed MIC values ranged from 4 to 128 microg/ml compared to the antibiotic drugs ethambutol (MIC ranged from 0.5 to 8 microg/ml) and isoniazid (MIC ranged from 1 to 4 microg/ml).     

Potential causes of arrested succession in Kibale National Park, Uganda: growth and mortality of seedlings

Recent studies suggest that regeneration following large-scale disturbance in Kibale National Park, Uganda, is slow or possibly arrested. Here, data is provided on the growth and mortality of seedlings in the forest understory, treefall gaps, and in large gaps that suggest that this pattern of arrested succession can be attributed partially to the fact that this East African community lacks aggressive colonizing tree species. Growth and mortality rates were contrasted for seedlings of six tree species planted in the understory, small gaps, and large gaps for 36 months. Data suggest that species are adapted to gaps of particular sizes. For example, Uvariopsis congensis grew faster in the understory than in small gaps, whereas Warburgia ugandensis had the lowest mortality rate and highest growth rate in large gaps. Seedlings (n=170) of 15 species were transplanted to assess the response of the tree community to large gap conditions. The limited survival of seedlings in large gaps relative to the understory suggests that only a small proportion of the tree community in this forest regenerates best in gaps larger than those created by the collapse of a single tree. These findings differ from a number of studies conducted in other geographical regions, and suggests that tree communities differ with respect to the proportion of tree species adapted to gaps of particular sizes. This may relate to variation among regions in their history of disturbance and thus frequency of gap formation, size of gaps, and the duration of periods of release. Such variation could imply the existence of a corresponding pattern among tropical forests of differential vulnerability to human disturbance, which tends to create many large gaps.     

Double jeopardy: bark harvest for malaria treatment and poor regeneration threaten tree population in a tropical forest of Uganda

Several forest plants known to supply medicine are under pressure worldwide. We carried out a study of four tree species (Warburgia ugandensis Sprague, Fleroya rubrostipulata (K.Schum.) Y.F.Deng, Syzygium guineense DC. and Zanthoxylum chalybeum Engl.) that are highly demanded for malaria treatment. The study was undertaken between 2006 and 2009 in the Sango Bay Forest Reserve, southern Uganda. The aim was to determine the conservation status of trees targeted for malaria treatment. We assessed the level of damage inflicted on trees during harvesting of medicinal parts and determined the population density of target species in the forest. We used 95 plots established along nineteen transects. Survival of the most preferred species, F. rubrostipulata, is of particular concern as its population suffered from a combination of extensive damage due to poor methods of harvesting and poor regeneration. The density of trees with diameter ≥5 cm differed between species and sites, demonstrating different recruitment and survival strategies. Management of Protected Areas should augment strategies to monitor the legal and illegal harvest of medicinal plants, by adopting low impact harvesting methods, and designating the temporal and spatial patterns of harvesting. This might reduce tree damage and mortality.     

An efficient callus-based in vitro regeneration protocol for Warburgia Ugandensis Sprague,an important medicinal plant in Africa

Warburgia ugandensis Sprague is a woody species in the family Canellaceae and an important source of medicines in Africa. Natural propagation of W. ugandensis is problematic due to its recalcitrant seeds and lack of an efficient in vitro regeneration system for this species. This study describes an efficient regeneration protocol. Petiole bases and shoot tips were used as explants. Callus tissue developed when the explants were cultured on Murashige and Skoog medium containing 30 g L⁻¹ sucrose and 7 g L⁻¹ agar (MS30 medium), supplemented with 1.0 mg L⁻¹ indole-3-butyric acid (IBA), 1.6 mg L⁻¹ 6-benzylaminopurine (BA), and 0.1 mg L⁻¹ thidiazuron (TDZ). Adventitious buds were efficiently induced from the callus when the MS30 medium was supplemented with 0.8 mg L⁻¹ BA and 0.2 mg L⁻¹ IBA. Root induction occurred within 7–10 d on half-strength MS30 medium supplemented with 0.8–1.0 mg L⁻¹ 1-napthalene acetic acid (NAA), 0.2 mg L⁻¹ IBA, and 0.03% (w/v) activated charcoal (AC). Roots were followed by root elongation on the same medium but lacking NAA and IBA. Approximately 50% of the plantlets cultured produced roots, while more than 80% of the plantlets survived and successfully grew to maturity.

     

Bioassay-guided isolation of three anthelmintic compounds from Warburgia ugandensis Sprague subspecies ugandensis, and the mechanism of action of polygodial

Parasitic helminths continue to pose problems in human and veterinary medicine, as well as in agriculture. Resistance to current anthelmintics has prompted the search for new drugs. Anthelmintic metabolites from medicinal plants could be good anthelmintic drug candidates. However, the compounds active against nematodes have not been identified in most medicinal plants with anthelmintic activity. In this study, we aimed to identify the active compounds against helminths in Warburgia ugandensis Sprague subspecies ugandensis (Canellaceae) and study the underlying mechanism of action. A bioassay-guided isolation of anthelmintic compounds from the plant was performed using a Caenorhabditis elegans (C. elegans) test model with a WMicrotracker instrument to monitor motility. Three active compounds were purified and identified by nuclear magnetic resonance and high resolution MS: warburganal (IC₅₀: 28.2?±?8.6?μM), polygodial (IC₅₀: 13.1?±?5.3?μM) and alpha-linolenic acid (ALA, IC₅₀: 70.1?±?17.5?μM). A checkerboard assay for warburganal and ALA as well as polygodial and ALA showed a fractional inhibitory concentration index of 0.41 and 0.37, respectively, suggesting that polygodial and ALA, as well as warburganal and ALA, have a synergistic effect against nematodes. A preliminary structure–activity relationship study for polygodial showed that the α,β-unsaturated 1,4-dialdehyde structural motif is essential for the potent activity. None of a panel of C. elegans mutant strains, resistant against major anthelmintic drug classes, showed significant resistance to polygodial, implying that polygodial may block C. elegans motility through a mechanism which differs from that of currently marketed drugs. Further measurements showed that polygodial inhibits mitochondrial ATP synthesis of C. elegans in a dose-dependent manner (IC₅₀: 1.8?±?1.0?μM). Therefore, we believe that the underlying mechanism of action of polygodial is probably inhibition of mitochondrial ATP synthesis. In conclusion, polygodial could be a promising anthelmintic drug candidate worth considering for further development.     

An integrated assessment of wild vegetable resources in Inner Mongolian Autonomous Region, China

Mwingi District lies within the Kenyan Arid and Semiarid lands (ASALs) in Eastern Province. Although some ethnobotanical surveys have been undertaken in some arid and semiarid areas of Kenya, limited studies have documented priority medicinal plants as well as local people's awareness of conservation needs of these plants. This study sought to establish the priority traditional medicinal plants used for human, livestock healthcare, and those used for protecting stored grains against pest infestation in Mwingi district. Further, the status of knowledge among the local people on the threat and conservation status of important medicinal species was documented. This study identified 18 species which were regarded as priority traditional medicinal plants for human health. In terms of priority, 8 were classified as moderate, 6 high, while 4 were ranked highest priority species. These four species are Albizia amara (Roxb.) Boiv. (Mimosacaeae), Aloe secundiflora (Engl. (Aloaceae), Acalypha fruticosa Forssk. (Euphorbiaceae) and Salvadora persica L. (Salvadoraceae). In regard to medicinal plants used for ethnoveterinary purposes, eleven species were identified while seven species were reported as being important for obtaining natural products or concoctions used for stored grain preservation especially against weevils. The data obtained revealed that there were new records of priority medicinal plants which had not been documented as priority species in the past. Results on conservation status of these plants showed that more than 80% of the respondents were unaware that wild medicinal plants were declining, and, consequently, few of them have any domesticated species. Some of the species that have been conserved on farm or deliberately allowed to persist when wild habitats are converted into agricultural lands include: Croton megalocarpus Hutch., Aloe secundiflora, Azadirachta indica A. Juss., Warburgia ugandensis Sprague, Ricinus communis L. and Terminalia brownie Fresen. A small proportion of the respondents however, were aware of the threats facing medicnal plants. Some of the plants reported as declining include, Solanum renschii Vatke (Solanaceae), Populus ilicifolia (Engl.) Rouleau (Salicaceae), Strychnos henningsii Gilg (Loganiaceae) and Rumex usambarensis (Dammer) Dammer (Polygonaceae). Considering the low level of understanding of conservation concerns for these species, there is need therefore, to build capacity among the local communities in this area particularly in regard to sustainable use of natural resources, conservation methods as well as domestication processes.     

The potential of South African plants in the development of new medicinal products

Southern Africa is an important focal point of botanical and cultural diversity but only a few plant species have hitherto become fully commercialised as medicinal products. In recent years there has been an upsurge in research and development activity, resulting in several new products and new crops. In this review, more than 90 of the best-known and most promising indigenous South African plants are listed and subjectively evaluated in the context of their potential for commercialisation as medicinal products for a variety of applications. The history of product development relating to the following species is briefly discussed and the plants and some of their products are illustrated: Agathosma betulina (buchu), Aloe ferox (bitter aloe), Artemisia afra (African wormwood), Aspalathus linearis (rooibos tea), Bulbine frutescens (burn jelly plant); Cyclopia genistoides (honeybush tea), Harpagophytum procumbens (devil's claw), Hoodia gordonii (hoodia, ghaap), Hypoxis hemerocallidea (“African potato”), Lippia javanica (fever tea), Mesembryanthemum tortuosum (= Sceletium tortuosum) (kanna, kougoed), Pelargonium sidoides (“Umckaloabo”), Siphonochilus aethiopicus (African ginger), Sutherlandia frutescens (= Lessertia frutescens) (cancer bush), Warburgia salutaris (pepperbark tree) and Xysmalobium undulatum (“Uzara”). The main factors that are apparently responsible for failure or success will be highlighted, especially the importance of marketing strategy, proof of concept and barriers to market entry.     

The impact of commercial harvesting on Warburgia salutaris (‘pepper-bark tree’) in Mpumalanga, South Africa

Commercialisation often increases the difficulty in managing harvested plant populations sustainably. The bark of the popular medicinal species, Warburgia salutaris (Bertol.f.) Chiov. (Canellaceae) (pepper-bark tree), is widely traded throughout southern Africa. The impact of commercial harvesting on this Red Data species was assessed by comparing commercially harvested populations with populations growing on private land or in protected areas (termed protected populations) in Mpumalanga and Limpopo Province, South Africa. The basal diameters and heights of stems in commercially harvested populations were significantly lower than those of the protected populations. The density of young/small plants was low in all populations. W. salutaris is usually resilient to high levels of bark harvesting. In this study, 75% of heavily harvested stems (>10% of the stem below 2 m) coppiced (resprouted). However, individuals that had been affected by regular fires, or repeatedly harvested, appeared prone to a fungal disease and had high percentage mortality. The populations occurring on private land appeared the most vigorous. Habitat in one protected area had been reduced through the construction of a dam. In another, small W. salutaris populations exhibited a shrubby growth form, probably due to frequent fires. Our current knowledge for this species supports a global IUCN status of EN A4acd. Plant conservation needs to become a higher priority both within and outside protected areas. Commercially harvested populations should be better managed through improved harvesting techniques and monitoring. Cultivation levels urgently need to be increased. Further research should be conducted on factors limiting regeneration, including the most appropriate fire regime.     

Safety of Prunus africana and Warburgia ugandensis in asthma treatment

The aim of the study was to determine the possible cytotoxicity of the aqueous stem bark extracts of Prunus africana and Warburgia ugandensis to Vero E6 cells and acute toxicity in BALB/c mice. Despite being some of the most popular medicinal plants used in Africa, little is known about the safety. In-vitro cytotoxicity tests on Vero E6 cells were investigated using MTT assay to assess the safety of the two plant extracts. Vero E6 cells on growing to confluence were incubated with different drug concentrations for 48 h for the drug to take effect. Viability of the cells was measured by a scanning multiwell spectrophotometer, color intensity being equivalent to viable cells which reduce MTT to soluble formazan crystals. This was done by determining the CC₅₀ of the extracts, CC₅₀ being the concentration of the dose of the compound/extract that kills 50% of the cells. In acute toxicity a total of 55 mice were used. Mice were divided into eleven groups of 5 mice, one group served as negative control and ten groups received oral gavage doses at 500, 889.56, 1581.6, 2812.15 or 5000 mg/kg body weight once. Mortality and other signs of toxicity were recorded within 24 h and the weights of the surviving mice taken for 14 days thereafter. P. africana had CC₅₀ of 104.08 μg/ml while W. ugandensis had CC₅₀ > 250 μg/ml and both were classified as not cytotoxic. There was no mortality observed in groups of mice that received P. africana extracts at 500 and 889.56 mg/kg body weight. There was 20%, 60% and 100% mortality observed within 24 h for mice that received P. africana extracts at 1581.64, 2812.15 and 5000 mg/kg body weight respectively. Lethal dose (LD₅₀) for P. africana was 2201.207 mg/kg body weight. W. ugandensis extracts had no mortality recorded in all dose levels and the LD₅₀ was > 5000 mg/kg body weight. The weights of mice that survived the entire 14 days in all groups increased and were not significantly different from that of controls p > 0.05. From the in vitro and in vivo studies, the two extracts were safe to use. Though with their customary value among many Kenyan communities in management of asthma among other ailments there is a need for further validation of any anti-asthmatic properties and responsible chemical compounds to augment the findings.     

Crystal structure, detonation performance, and thermal stability of a new polynitro cage compound: 2, 4, 6, 8, 10, 12, 13, 14, 15-nonanitro-2, 4, 6, 8, 10, 12, 13, 14, 15-nonaazaheptacyclo [5.5.1.13, 11. 15, 9] pentadecane

A new polynitro cage compound 2, 4, 6, 8, 10, 12, 13, 14, 15-nonanitro-2, 4, 6, 8, 10, 12, 13, 14, 15-nonaazaheptcyclo [5.5.1.1(3,11).1(5,9)] pentadecane (NNNAHP) was designed in the present work. Its molecular structure was optimized at the B3LYP/6-31 G(d,p) level of density functional theory (DFT) and crystal structure was predicted using the Compass and Dreiding force fields and refined by DFT GGA-RPBE method. The obtained crystal structure of NNNAHP belongs to the P-1 space group and the lattice parameters are a = 9.99 ?, b = 10.78 ?, c = 9.99 ?, α = 90.01°, β = 120.01°, γ = 90.00°, and Z = 2, respectively. Based on the optimized crystal structure, the band gap, density of state, thermodynamic properties, infrared spectrum, strain energy, detonation characteristics, and thermal stability were predicted. Calculation results show that NNNAHP has detonation properties close to those of CL-20 and is a high energy density compound with moderate stability.     

Simultaneous measurement of the trace elements Al, As, B, Be, Cd, Co, Cu, Fe, Li, Mn, Mo, Ni, Rb, Se, Sr, and Zn in human serum and their reference ranges by ICP-MS

The goal of this article was to establish reference ranges of the concentration of trace elements in human serum and to compare these results with those reported by other authors. We describe the sample preparation and measurement conditions that allow the rapid, precise, and accurate determination of Al, As, B, Be, Cd, Co, Cu, Fe, Li, Mn, Mo, Ni, Rb, Se, Sr, and Zn in human serum samples (n=110) by inductively coupled plasma-mass spectrometry (ICP-MS). Accuracy and precision were determined by analyzing three reconstituted reference serum samples by comparison with other methods and by the standard addition procedure. The advantages of the ICP-MS method include short time of analysis of the elements mentioned, low detection limit, high precision, and high accuracy. Disadventages include a high risk of contamination due to the presence of some of the elements of interest in the environment, the relatively delicate sample handling, and the high cost of the equipment.